1. Field of the Invention
The present invention relates to a hematopoietic stem cell identification probe, and a method and a kit using a hematopoietic stem cell identification probe.
2. Description of the Related Art
A hematopoietic stem cell is a cell that has multipotency of differentiating into any of all types of blood cells including leucocytes (such as a granulocyte, a lymphocyte, a monocyte and a macrophage), a erythrocyte, a platelet, a mast cell and a denderitic cell, and has replication competence. As a treatment for diseases causing a damage in the hematopoietic function, for example, hematological cancers such as leukaemia, malignant lymphoma and multiple myeloma, or aplastic anemia, transplantation of hematopoietic stem cells accompanied by chemotherapy and radiation therapy has been established.
For performing the transplantation of hematopoietic stem cells, it is necessary to efficiently collect cells including hematopoietic stem cells from umbilical cord blood, bone marrow, peripheral blood or the like and to identify, segregate and enrich the collected cells. Furthermore, use of hematopoietic stem cells of mammals other than a human (such as a mouse and a rat) has been earnestly studied as a model for clarifying mechanism of differentiating and mechanism of maintaining an undifferentiated state of somatic stem cells, and also in such studies, there is a demand for an efficient method for identifying, segregating and enriching hematopoietic stem cells.
As a method for segregating hematopoietic stem cells, a method in which a monocyte component is taken out by using a blood component centrifuge and surfaces of hematopoietic stem cells are labeled with magnetic beads so as to segregate the hematopoietic stem cells from unlabeled cells, or a method using a cell sorter is employed.
If a cell sorter is used, a method of selectively sorting cells labeled with a cell surface marker and a method of sorting cells having a dye excretion property are known, and cells can be highly accurately segregated by such a method.
As for fractions obtained by using a cell sorter with a cell surface marker used, it has been clarified that a fraction having a pattern of a cell surface antigen expression of c-Kit positive, Sca-1 positive and lineage marker negative (a KSL fraction) and, with respect to those obtained from a mouse, a fraction further being CD34 negative or a CD34 negative-KSL fraction and a KSL-SP fraction contain hematopoietic stem cells in a high concentration, and these fractions are frequently used in the same sense as hematopoietic stem cells. C-Kit is known to be expressed in all hematopoietic precursor cells having multipotency similar to that of hematopoietic stem cells and can be used as a simple marker for hematopoietic stem cells and undifferentiated hematopoietic cells including hematopoietic precursor cells. Furthermore, it has been reported that hematopoietic stem cells are enriched in CD150 positive and CD48 negative fractions, and such a fraction is used singly or together with a CD34 negative-KSL fraction.
Furthermore, a method using JAM-1 as a marker (Japanese Patent Application Laid-Open No. 2004-242513), a method using Robo-4 protein as a maker (WO2007/010586) and a method using a marker for hyaluronic acid present on a cell surface (Japanese Patent Application Laid-Open No. 2009-232853) have been disclosed.
On the other hand, as a method using a dye, a method of enriching a SP (side population) of cells specifically excreting Hoechst 33342 bound to DNA is widely known (Goodell M A et al., J Exp Med. 1996; 183, 1797-1806). Moreover, it has been disclosed that Rhodamine 123 is excreted by hematopoietic stem cells (Wolf, N S et al., Exp Hematol. 1993; 21, 614-622).
If a hematopoietic stem cell enriched cell population is easily identified and separated, not only it is extremely useful in transplantation of enriched/separated hematopoietic stem cells for reproducing the hematogenous function of a patient having been given a medical treatment causing a damage in hematopoiesis, such as a chemotherapy for a cancer, but also it makes it possible to perform image analysis of hematopoietic stem cells, to identify a bioactive substance affecting the behavior of hematopoietic stem cells, and the like.
The methods of segregating/enriching hematopoietic stem cells using a cell surface marker disclosed in Japanese Patent Application Laid-Open No. 2004-242513, WO2007/010586, Japanese Patent Application Laid-Open No. 2009-232853 and the like had, however, problems, for example, in which it costs much because a plurality of expensive monoclonal antibodies and the like are used in combination, the functions of segregated cells may be inhibited by an antibody, and a blocking operation for preventing non-specific adsorption of an antibody may be required in some cases.
On the other hand, the method using a dye is superior in a point where a reagent is available inexpensively as compared with an antibody, but it is necessary to excite Hoechst 33342 at a wavelength in the vicinity of 350 nm. Although an ultraviolet laser is necessary for coping with this wavelength, an ultraviolet laser is too expensive to be provided on a general cell sorter apparatus, and therefore, Hoechst 33342 could not be widely used. Moreover, since Hoechst 33342 is a DNA-binding dye, there was a problem in which Hoechst 33342 should be carefully handled because there is a risk of mutagenicity. The Rhodamine 123 disclosed by Wolf, N S et al., in Exp Hematol. 1993; 21, 614-622 is excited at a wavelength in the vicinity of 500 nm and hence the fluorescence can be detected by an inexpensive analyzer. The Rhodamine 123 is, however, excreted by many hematopoietic cells other than hematopoietic stem cells, and hence is less efficiently used for enrichment of hematopoietic stem cells.